The global increase in tuberculosis concomitant with the spread of HIV infection has led to renewed interest in investigating the virulence determinants of Mycobacteria tuberculosis. The goal of this research is to study the interaction of pathogenic Mycobacteria with the host cell, to characterize cellular and bacterial components required for bacterial entry into and survival within host cells, and to identify bacterial components required for long term survival in the host. We are using Mycobacterium marinum and M. ulcerans, both of which cause persistent disease in humans, as models for studying virulence factors in M. tuberculosis. Taxonomic studies undertaken in the laboratory have shown that M. marinum and M. ulcerans are more closely related to M. tuberculosis than any Mycobacterial species except for M. Bovis. Using cultured cell lines we have shown that M. marinum replicates within macrophages, epithelial cells and fibroblasts. Using confocal microscopy we have found that intracellular M. marinum, Like M. tuberculosis resides in a unique vesicle which does not fuse with lysosomes. We have isolated an M. marinum mutant which enters but does not persist within macrophages. This mutant lacks a phospholipase and phospholipase activity present in the parental strain and also present in M. tuberculosis. Compared to the wild type parental strain in a guinea pig and frog model, this mutant is severely attenuated. We have begun to identify genes involved in the attenuated phenotype. The identification of these genes should yield insight into the pathogenesis of tuberculosis. Like M. tuberculosis, M. ulcerans causes a persistent infection in humans, Buruli ulcer. Organisms secrete a cytotoxin which is likely to be responsible for several unique features of the infection including the lack of a normal inflammatory response to the presence of the bacteria. In characterizing the interaction of M. ulcerans with cultured cell lines we have found that M. ulcerans do not adhere to cells and are poorly phagocytosed. Sterile filtrate from M. ulcerans profoundly alters the biology of the host cell. Although cells are not killed upon exposure to culture filtrate, they undergo cell cycle arrest. In addition, interaction of host cells with the cytotoxin leads to suppression of IL-1alpha production which may be responsible for the absence of neutrophils in necrotic lesions. This research may lead to the development of useful pharmaceuticals.